The blue dye indigo is one of the oldest dyestuffs known to man. Its use as a textile dye dates back to at least 2000 BC. Until the late 1800s indigo, or indigotin, was principally obtained from plants of the genus Indigofera, which range widely in Africa, Asia, the East Indies and South America. As the industrial revolution swept through Europe and North America in the 1800s, demand for the dye's brilliant blue color lead to its development as one of the main articles of trade between Europe and the Far East. In 1883 Alfred von Baeyer identified the formula of indigo: C.sub.16 H.sub.10 N.sub.2 O.sub.2. In 1887 the first commercial chemical manufacturing process for indigo was developed. This process, still in use today, involves the fusion of sodium phenylglycinate in a mixture of caustic soda and sodamide to produce indoxyl. The process' final product, indoxyl, oxidized spontaneously to indigo by exposure to air.
Current commercial chemical processes for manufacturing indigo result in the generation of significant quantities of toxic waste products. Obviously, a method whereby indigo may be produced without the generation of toxic by-products is desirable. One such method which results in less toxic by-product generation involves indigo biosynthesis by microorganisms.
Ensley et al. [(1983) Science 222:167-169] found that a DNA fragment from a transmissible plasmid isolated from the soil bacterium Pseudomonas putida enabled Escherichia coli stably transformed with a plasmid harboring the fragment to synthesize indigo in the presence of indole or tryptophan. Ensley et al. postulated that indole, added either as a media supplement or produced as a result of enzymatic tryptophan catabolism, was converted to cis-indole-2,3-dihydrodiol and indoxyl by the previously identified multi-component enzyme naphthalene dioxygenase (NDO) encoded by the P. putida DNA fragment. The indoxyl so produced was then oxidized to indigo upon exposure to air. The dioxygenase described by Ensley et al. is a preferred oxygenase useful in the production of indigo as further described herein.
NDO had previously been found to catalyze the oxidation of the aromatic hydrocarbon naphthalene to (+)-cis-(1 R,2S)-dihydroxy-1,2-dihydronaphthalene [Ensley et al. (1982) J. Bacteriol. 149:948-954]. U.S. Pat. No. 4,520,103, incorporated by reference, describes the microbial production of indigo from indole by an aromatic dioxygenase enzyme such as NDO. The NDO enzyme is comprised of multiple components: a reductase polypeptide (Rd, molecular weight of approximately 37,000 daltons (37 kD)); an iron-sulfur ferredoxin polypeptide (Fd, molecular weight of approximately 13 kD); and a terminal oxygenase iron-sulfur protein (ISP). ISP itself is comprised of four subunits having an .alpha..sub.2.beta..sub.2 subunit structure (approximate subunit molecular weights: .alpha., 55 kD; .beta.,21 kD). ISP is known to bind naphthalene, and in the presence of NADH, Rd, Fd and oxygen, to oxidize it to cis-naphthalene-dihydrodiol. Fd is believed to be the rate-limiting polypeptide in this naphthalene oxidation catalysis, (see U.S. Pat. No. 5,173,425, incorporated herein by reference, for a thorough discussion of the various NDO subunits and ways to improve them for purposes of indigo biosynthesis).
In addition, aromatic dioxygenases other than NDO may also be useful in the biosynthetic production of indigo, for example, a toluene monooxygenase (TMO) such as that from Pseudomonas (P. mendocina) capable of degrading toluene was also able to produce indigo when the culture medium was supplemented with indole. For details, see U.S. Pat. No. 5,017,495, incorporated herein by reference. In principle, any enzyme capable of introducing a hydroxyl moiety into the 3-position of indole to give indoxyl is a candidate for use in the biosynthetic production of indigo.
It has also long been known that microorganisms contain biosynthetic pathways for the production of all 20 essential amino acids, including the aromatic amino acid L-tryptophan. The de novo synthesis of aromatic amino acids (phenylalanine, tryptophan and tyrosine) share a common pathway up through the formation of chorismic acid. After chorismic acid synthesis, specific pathways for each of the three aromatic amino acids are employed to complete their synthesis.
Bacterial biosynthesis of tryptophan from chorismic acid (specifically in E. coli) is under the control of the tryptophan (trp) operon. The (trp) operon, comprised of regulatory regions and several structural genes, has been extensively studied because of its complex and coordinated regulatory systems. The regulatory and structural organization of the E coli trp operon, along with the catalytic activities encoded by the structural genes of the operon, appear in FIG. 1 of PCT/US93/09433, incorporated herein by reference. PCT/US93/09433 describes improvements in the intracellular production of indole, specifically as it relates to the conversion of indole-3'-glycerol-phosphate (InGP), in conjunction with L-serine, to L-tryptophan. The reaction is catalyzed by the multi-subunit enzyme tryptophan synthase. During the reaction, indole is produced as an intermediate.
However, the indole is very rapidly combined with L-serine in a stoichiometric fashion to produce L-tryptophan. Thus, no free indole is produced as a result of this InGP plus L-serine conversion to tryptophan.
However, Yanofsky et al. [(1958) Biochim. Biophys. Acta. 28:640-641] identified a tryptophan synthase mutant which led to the accumulation of indole. This particular tryptophan synthase mutant, however, was subject to spontaneous reversion to the wild-type phenotype, as the mutation resulted from a single nucleotide base pair change in the gene coding for the .beta. subunit of tryptophan synthase.
PCT/US93/09433 describes a method for creating stable tryptophan synthase mutants capable of yielding high levels of intracellular indole. When such indole accumulating tryptophan synthase mutants express an aromatic dioxygenase enzyme like NDO, the accumulated indole may be converted to indoxyl, which can then be oxidized to indigo by molecular oxygen. Thus, through the commercial application of recombinant DNA technology, by the overexpression of a modified trp operon capable of continuously producing indole and an oxygenase enzyme capable of simultaneous conversion of indole to indoxyl, indigo can be produced from a renewable raw material such as glucose.
In shake flask studies applicants have determined that during the synthesis of indigo from indole, low levels of other compounds or by-products accumulate in the culture supernatant. One of these by-products, isatin (indole 2,3-dione), has been found to inhibit the oxygenase (i.e., NDO) activity in the production strain and, consequently, reduces overall indigo production; thus, isatin is undesirable. In addition to the by-product isatin, indirubin, a red dye material derived from isatin, may be produced during this biosynthetic indigo production process. The by-product isatin is believed to reduce the productivity of the production strain, while the by-product indirubin is believed to cause undesirable dyeing characteristics to microbially produced indigo which is expressed as a red cast on cloth dyed with indirubin-tainted microbially produced indigo.
Because the production in shake flasks of one or more of these by-products may either reduce the productivity of this production strain and/or cause undesirable characteristics of the indigo produced therefrom, an object of the present invention is to reduce the buildup of isatin or remove such isatin formed as a by-product of biosynthesis of indigo in microbial cells. Removal of isatin will potentially enhance the overall production of indigo in a fermentor and reduce or prevent the accumulation of indirubin. One method to reduce the buildup of isatin or remove such isatin, as detailed herein, relates to the isolation, cloning, sequencing and expression in indigo-producing host strains of a gene encoding an enzyme having isatin-removing activity. Preferably the enzyme is an isatin hydrolase, an enzyme capable of degrading isatin; however, any method to remove or inhibit isatin formation is contemplated by the present invention. Thus, another aspect of the present invention is the enhanced production of biosynthetic indigo by reducing the buildup or removing accumulated isatin through means, including, but not limited to, enzymatic conversion of the isatin by contacting it with an isatin-removing enzyme such as an isatin hydrolase, general base catalyzed chemical conversion of the isatin at appropriate temperature and pH, or through adsorption of the isatin to carbon or a suitable resin. These aspects of the invention are detailed below.
Definition of Terms
The following terms will be understood as defined herein unless otherwise stated. Such definitions include without recitation those meanings associated with these terms known to those skilled in the art.
Tryptophan pathway genes useful in securing biosynthetic indole accumulation include a trp operon, isolated from a microorganism as a purified DNA molecule that encodes an enzymatic pathway capable of directing the biosynthesis of L-tryptophan from chorismic acid. (A.J. Pittard (1987) Biosynthesis of Aromatic Amino Acids in Escherichia coli and Salmonella typhimurium, F. C. Neidhardt, ed., American Society for Microbiology, publisher, pp. 368-394.) Indole accumulation is enabled by modification of one or more of the pathway's structural elements and/or regulatory regions. This modified trp operon may then be introduced into a suitable host such as a microorganism, plant tissue culture system or other suitable expression system. It should be noted that the term "indole accumulation" does not necessarily indicate that indole actually accumulates intracellularly. Instead, this term can indicate that there is an increased flux of carbon to indole and indole is made available as a substrate for intracellular catalytic reactions such as indoxyl formation and other than the formation of L-tryptophan. In the context of this invention, the "accumulated" indole may be consumed in the conversion of indole to indoxyl by an oxygenase such as the aromatic dioxygenase NDO, or an aromatic monooxygenase such as TMO, or it may actually build up intracellularly and extracellularly, as would be the case when the desired end product is indole or one of its derivatives.
A suitable host microorganism or host cell is an autonomous single-celled organism useful for microbial indole and/or indigo production and includes both eucaryotic and procaryotic microorganisms. Such host microorganism contains all DNA, either endogenous or exogenous, required for the production of indole, indoxyl and/or indigo, either from glucose or as a bioconversion from tryptophan. Useful eucaryotes include organisms like yeast and fungi or plants. Prokaryotes useful in the present invention include, but are not limited to, bacteria such as E. coli, P. putida and Salmonella typhimurium.
Biosynthetic conversion of indole to indigo is meant to include indoxyl oxidation to indigo mediated by molecular oxygen or air.
A DNA fragment, as used herein, may encode regulatory and/or structural genetic information. A DNA fragment useful in the instant invention shall also include: nucleic acid molecules encoding sequences complementary to those provided; nucleic acid molecules (DNA or RNA) which hybridize under stringent conditions to those molecules that are provided; or those nucleic acid molecules that, but for the degeneracy of the genetic code, would hybridize to the molecules provided or their complementary strands. "Stringent" hybridization conditions are those that minimize formation of double stranded nucleic acid hybrids from non-complementary or mismatched single stranded nucleic acids. In addition, hybridization stringency may be effected by the various components of the hybridization reaction, including salt concentration, the presence or absence of formamide, the nucleotide composition of the nucleic acid molecules, etc. The nucleic acid molecules useful in the present invention may be either naturally or synthetically derived.
An "exogenous" DNA fragment is one that has been introduced into the host microorganism by any process such as transformation, transfection, transduction, conjugation, electroporation, etc. Additionally, it should be noted that it is possible that the host cell into which the "exogenous" DNA fragment has been inserted may itself also naturally harbor molecules encoding the same or similar sequences. For example, when E. coli is used in this invention as the host strain, it is recognized that normally the host naturally contains, on its chromosome, a trp operon capable of directing the synthesis of L-tryptophan from chorismic acid under conditions enabling trp operon expression. A molecule such as this is referred to as an "endogenous" DNA molecule.
A stably transformed microorganism is one that has had one or more exogenous DNA fragments introduced such that the introduced molecules are maintained, replicated and segregated in a growing culture. Stable transformation may be due to multiple or single chromosomal integration(s) or by extrachromosomal element(s) such as a plasmid vector(s). A plasmid vector is capable of directing the expression of polypeptides encoded by particular DNA fragments. Expression may be constitutive or regulated by inducible (or repressible) promoters that enable high levels of transcription of functionally associated DNA fragments encoding specific polypeptides such as the structural genes of a trp operon modified as described herein.
An "isatin-removing enzyme," as used herein, is any enzyme which comprises activity resulting in the inhibition, removal, inactivation, degradation, hydrolysis or binding (sequestering) of isatin, whether such enzyme causes the formation of isatic acid or any other derivative of isatin. A preferred isatin-removing enzyme useful in the present invention is an isatin hydrolase such as the hydrolase isolated from Pseudomonas putida (WW2) herein.
Regardless of the exact mechanism utilized for expression of enzymes necessary for the microbial production of indole, indoxyl and/or indigo, it is contemplated that such expression will typically be effected or mediated by the transfer of recombinant genetic elements into the host cell. Genetic elements as herein defined include nucleic acids (generally DNA or RNA) having expressible coding sequences for products such as proteins, specifically enzymes, apoproteins or antisense RNA, which express or regulate expression of relevant enzymes (i.e., isatin hydrolase, tryptophan synthase, NDO, etc.). The expressed proteins can function as enzymes, repress or derepress enzyme activity or control expression of enzymes. Recombinant DNA encoding these expressible sequences can be either chromosomal (integrated into the host cell chromosome by, for example, homologous recombination) or extrachromosomal (for example, carried by one or more plasmids, cosmids and other vectors capable of effecting the targeted transformation). It is understood that the recombinant DNA utilized for transforming the host cell in accordance with this invention can include, in addition to structural genes and transcription factors, expression control sequences, including promoters, repressors and enhancers, that act to control expression or derepression of coding sequences for proteins, apoproteins or antisense RNA. For example, such control sequences can be inserted into wild-type host cells to promote overexpression of selected enzymes already encoded in the host cell genome, or alternatively they can be used to control synthesis of extrachromosomally encoded enzymes.
The recombinant DNA can be introduced into the host cell by any means, including, but not limited to, plasmids, cosmids, phages, yeast artificial chromosomes or other vectors that mediate transfer of genetic elements into a host cell. These vectors can include an origin or replication, along with cis-acting control elements that control replication of the vector and the genetic elements carried by the vector. Selectable markers can be present n the vector to aid in the identification of host cells into which genetic elements have been introduced. Exemplary of such selectable markers are genes that confer resistance to particular antibiotics such as tetracycline, ampicillin, chloramphenicol, kanamycin or neomycin.
A means for introducing genetic elements into a host cell utilizes an extrachromosomal multi-copy plasmid vector into which genetic elements in accordance with the present invention have been inserted. Plasmid borne introduction of the genetic element into host cells involves an initial cleaving of a plasmid vector with a restriction enzyme, followed by ligation of the plasmid and genetic elements encoding for the targeted enzyme species in accordance with the invention. Upon recircularization of the ligated recombinant plasmid, infection (e.g., packaging in phage lambda) or other mechanism for plasmid transfer (e.g., electroporation, microinjection, etc.) is utilized to transfer the plasmid into the host cell. Plasmids suitable for insertion of genetic elements into the host cell are well known to the skilled artisan.